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Methods for isolating human cardiac ventricular progenitor cells

Active Publication Date: 2018-05-31
PROCELLA THERAPEUTICS AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention relates to a method of isolating human cardiac ventricular progenitor cells (HVPs) from human pluripotent stem cells using negative selection of designated markers. The isolated HVPs can be used for in vivo tissue engineering and have a high degree of ventricular muscle cell differentiation. The use of negative selection ensures a rigorous definition of the HVP population and eliminates potential teratoma-causing cells. The method involves culturing the negative selection markers in a specific medium that promotes ventricular differentiation and expanding the cells to a clonal population.

Problems solved by technology

Although heart transplantation can be curative, the markedly limited availability of human heart organ donors has led to a widespread unmet clinical need for a renewable source of pure, mature and functional human ventricular muscle tissue (Segers, V. F. M. and Lee, R. J. (2008) Nature 451:937-942; Später, D. et al.
While human pluripotent stem cells hold great promise, a significant challenge has been the ability to move from simply differentiation of diverse cardiac cells to forming a larger scale pure 3D ventricular muscle tissue in vivo, which ultimately requires vascularization, assembly and alignment of an extracellular matrix, and maturation.
While the identification of Isl1 as a marker of cardiac progenitor cells was a significant advance, since Isl1 is an intracellular protein it is not a suitable marker for use in isolating large quantities of viable cells.

Method used

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  • Methods for isolating human cardiac ventricular progenitor cells
  • Methods for isolating human cardiac ventricular progenitor cells
  • Methods for isolating human cardiac ventricular progenitor cells

Examples

Experimental program
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Effect test

example 1

n of Human Isl1+ Cardiomyogenic Progenitor Cells by Modulation of Wnt Signaling in Human Pluripotent Stem Cells

[0223]Temporal modulation of canonical Wnt signaling has been shown to be sufficient to generate functional cardiomyocytes at high yield and purity from numerous hPSC lines (Lian, X. et al. (2012) Proc. Natl. Acad. Sci. USA 109:E1848-1857; Lian, X. et al. (2013) Nat. Protoc. 8:162-175). In this approach, Wnt / β-catenin signaling first is activated in the hPSCs, followed by an incubation period, followed by inhibition of Wnt / β-catenin signaling. In the originally published protocol, Wnt / β-catenin signaling activation was achieved by incubation with the Gsk3 inhibitor CHIR99021 (GSK-3 α, IC50=10 nM; GSK-3, β IC50=6.7 nM) and Wnt / β-catenin signaling inhibition was achieved by incubation with the Porcn inhibitor IWP2 (IC50=27 nM). Because we used Gsk3 inhibitor and Wnt production inhibitor for cardiac differentiation, this protocol was termed GiWi protocol. To improve the effici...

example 2

ation of Jagged 1 as a Cell Surface Marker of Cardiac Progenitor Cells

[0231]To profile the transcriptional changes that occur during the cardiac differentiation process at a genome-scale level, RNA sequencing (RNA-seq) was performed at different time points following differentiation to build cardiac development transcriptional landscapes. We performed RNA-seq experiments on day 0 to day 7 samples, as well as day 19 and day 35 samples (two independent biological replicates per time point). Two batches of RNA-seq (100 bp and 50 bp read length) were performed using the illumine Hiseq 2000 platform. In total, 20 samples were examined. Bowtie and Tophat were used to map our reads into a reference human genome (hg19) and we calculate each gene expression (annotation of the genes according to Refseq) using RPKM method (Reads per kilobase transcript per million reads). Differentiation of hPSCs to cardiomyocytes involves five major cell types: pluripotent stem cells (day 0), mesoderm progeni...

example 3

fferentiation of Isl1+Jag1+ Cardiac Progenitor Cells

[0240]To characterize the clonal differentiation potential of Isl1+Jag1+ cells, cardiomyogenic progenitor cells were generated by the culturing protocol described in Example 1, and one single Isl1+Jag1+ cell was seeded into one well of a Matrigel-coated 48-well plate. Cells were purified with antibody of Jag1 and then one single cell was seeded into one well. The single cells were then cultured for 3 weeks in Cardiac Progenitor Culture (CPC) medium (advanced DMEM / F12 supplemented with 2.5 mM GlutaMAX, 100 μg / ml Vitamin C, 20% Knockout Serum Replacement).

[0241]Immunostaining of the 3-week differentiation cell population was then performed with three antibodies: cardiac troponin I (cTn1) for cardiomyocytes, CD144 (VE-cadherin) for endothelial cells and smooth muscle actin (SMA) for smooth muscle cells. The results showed that the single cell-cultured, Isl1+Jag1+ cells gave rise to cTnI positive and SMA positive cells, but not VE-cadh...

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Abstract

The present invention provides methods for isolating human cardiac ventricular progenitor cells (HVPs), wherein cultures of day 5-7 cardiac progenitor cells are negatively selected for one or more first markers expressed on human pluripotent stem cells, such as TRA-1-60, to thereby isolate HVPs. The methods can further include positive selection for expression of a second marker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9. Large populations, including clonal populations, of isolated HVPs that are first marker negative / second marker positive are also provided. Methods of in vivo use of the HVPs for cardiac repair or to improve cardiac function are also provided. Methods of using the HVPs for cardiac toxicity screening of test compounds are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims priority to, and the benefit of, U.S. Provisional Application No. 62 / 427,569, filed on Nov. 29, 2016, the entire contents of which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Heart failure, predominantly caused by myocardial infarction, is the leading cause of death in both adults and children worldwide and is increasing exponentially worldwide (Bui, A. L. et al. (2011) Nat. Rev. Cardiol. 8:30-41). The disease is primarily driven by the loss of ventricular muscle that occurs during myocardial injury (Lin, Z. and Pu, W. T. (2014) Sci. Transl. Med. 6:239rv1) and is compounded by the negligible ability of the adult heart to mount a regenerative response (Bergmann, O. et al. (2009) Science 324:98-102; Senyo, S. E. et al. (2013) Nature 493:433-436). Although heart transplantation can be curative, the markedly limited availability of human heart organ donors has led to a widespread unmet clinical ...

Claims

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Application Information

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IPC IPC(8): C12N5/077A61K35/34G01N33/50
CPCC12N2513/00C12N2506/45C12N2501/599C12N2501/50C12N5/0657A61K35/34G01N33/5014G01N33/5061C12N2501/415C12N2501/42C12N2501/727C12N2506/02A61P9/00A61P9/10
Inventor CHIEN, KENNETH R.CLARKE, JONATHANLEHTINEN, MIIAFOO, KYLIELEUNG, CHUEN YAN
Owner PROCELLA THERAPEUTICS AB
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